A multiphasic approach to solve misidentification of Cutibacterium acnes as Atopobium vaginae during routine bacterial screening of platelet concentrates using the VITEK 2 system

Skin flora bacteria, such as Cutibacterium acnes , are the predominant contaminants of blood products used for transfusion. Platelet concentrates (PCs), a therapeutic product used to treat patients with platelet deficiencies, are stored at ambient temperature under agitation, providing ideal conditions for bacterial proliferation. At Canadian Blood Services, PCs are screened for microbial contamination using the automated BACT/ALERT culture system. Positive cultures are processed and contaminating organisms are identified using the VITEK 2 system. Over a period of approximately 2 years, several PC isolates were identified as Atopobium vaginae to a high level of confidence. However, since A. vaginae is associated with bacterial vaginosis and is not a common PC contaminant, a retrospective investigation revealed that in all cases C. acnes was misidentified as A. vaginae . Our investigation demonstrated that the media type used to grow PC bacterial isolates can have a significant impact on the results obtained on the VITEK 2 system. Furthermore, other identification methods such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALD-TOF MS) and PCR amplification of the 16S RNA gene were only partially successful in the identification of C. acnes . Therefore, our findings support a multiphasic approach when PC isolates are identified as A. vaginae by the VITEK 2 system for proper identification of C. acnes using macroscopic, microscopic and other biochemical analyses.


INTRODUCTION
Platelet concentrates (PCs) are manufactured by Canadian Blood Services to treat patients with bleeding disorders or platelet deficiencies. PCs are screened for bacterial contamination using the BACT/ALERT 3D system with aerobic (BPA) and anaerobic (BPN) culture bottles [1]. When a bottle flags positive, the contaminant is isolated by subculturing samples onto different agar types, including tryptic soy agar with 5 % sheep blood (TSAb), chocolate agar and anaerobic agar, such as CDC anaerobic blood agar with 5 % sheep blood (CDC) or OxyPRAS Plus (Brucella agar containing 5 % sheep blood and oxyrase) (Oxy), which are then incubated under different environmental conditions (aerobic, capnophilic and anaerobic, respectively). When growth is observed, a Gram stain is performed, and the isolates are identified using the VITEK 2 system and the appropriate VITEK 2 cards chosen based on the algorithm provided by the manufacturer. No other complementary tests are performed unless prompted by the VITEK 2 system. Data gathered at Canadian Blood Services between 2017-2019 indicate that the most isolated bacterial OPEN ACCESS contaminants of PCs are skin commensals such as Cutibacterium acnes, and staphylococcal species [1]. C. acnes, an anaerobic, aerotolerant, catalase-positive, Gram-positive, rod-shaped bacterium [2], is the most routinely isolated contaminant of PCs, accounting for approximately 70 % of bacterial species identified from positive BACT/ALERT cultures [1]. The VITEK 2 system was adopted for identification of blood product contaminants in 2019, prior to which, microbial identification at Canadian Blood Services was performed with the manual analytical profile index (API) identification system. In the 2 years following implementation of identification with the VITEK 2 system, there were several unprecedented instances of anaerobic bacterial isolates being identified as Atopobium vaginae to a high level of confidence (>95 %). Furthermore, several isolates could only be identified following a catalase test prompted by the VITEK 2 system, since it could not differentiate between A. vaginae and C. acnes.
A. vaginae, a member of the vaginal microflora, is a strict anaerobic, Gram-positive, catalase-negative, rod-shaped coccus [3], and is usually associated with bacterial vaginosis [4]. It should be noted, however, that there have been reports of A. vaginae being isolated from blood cultures from individuals experiencing transient bacteraemia [5], infective endocarditis [6] and sepsis [7]. Since A. vaginae was an unusual organism to be detected during screening of PCs obtained from healthy blood donors, a retrospective study was initiated to determine if these isolates may have been misidentified. The steps taken to ascertain the identities of these isolates have been described herein and provide recommendations on how to proceed when a PC isolate is identified as A. vaginae.

Bacterial strains
For the investigation, 8 catalase-positive PC isolates that were misidentified as A. vaginae were selected, together with 13 other isolates that were identified as C. acnes (see Table 1). The C. acnes ATCC 6919 strain and A. vaginae ATCC BAA-55 strain were used to evaluate microscopic and macroscopic morphology, and served as controls for VITEK identification and C. acnes 16S RNA PCR. Clostridium sordellii ATCC 9714 served as the control for API tests. Staphylococcus aureus ATCC 25923 and Streptococcus pyogenes ATCC 19615 were used as the positive and negative controls, respectively, for the catalase test. Clostridium septicum ATCC 12464 and Bacteriodes ovatus ATCC BAA-1296, which are quality control (QC) strains for the ANC card recommended by the VITEK 2 vendor, were used to perform weekly qualifications of the VITEK 2 system.

Agar media
Four agar media were used in this study. Three media, TSAb and CDC or Oxy, were used for the initial isolation of anaerobes during routine PC testing as per standard protocols at Canadian Blood Services. Columbia blood agar supplemented with 5 % sheep blood (CBA) was used to grow the QC controls for the anaerobic (ANC) VITEK 2 card as per the vendor's recommendations.

Assessing macroscopic and microscopic colony morphology
The ATCC strains of C. acnes (ATCC 6919) and A. vaginae (BAA-55) were grown on TSAb, CDC and CBA plates for 72 h, under anaerobiosis at 37 °C, and the colony morphology of the two species was assessed. Furthermore, smears were prepared, and Gram stain was performed to determine the microscopic differences between the two species. Additionally, a catalase test was performed as per the catalase test protocol described by the American Society of Microbiology [8]. Briefly, four well-isolated colonies of the ATCC C. acnes, A. vaginae and the catalase control strains grown on TSAb were picked using a sterile loop, ensuring that no agar was transferred, and were deposited on a clean, labelled microscope slide, to which a single drop of 3 % hydrogen peroxide was added, after which the slides were assessed for the production of effervescence.

Impact Statement
Microbial identification with automated systems based on biochemical reactions is common practice in clinical and industrial settings. These systems provide information about contaminants that aid decision making for patient treatment, or in the case of blood suppliers, provides information for blood product disposition, follow up of blood donors, and in some cases, follow up of transfusion patients. It is therefore very important to have accurate microbial identification results. To our knowledge, we report an issue so far not yet documented in literature concerning the misidentification of the skin flora bacterium Cutibacterium acnes as Atopobium vaginae, a microorganism involved in bacterial vaginosis. We showed that the automated VITEK identification system misidentified C. acnes on several occasions and that the most plausible cause for the unexpected results was the culture media used to isolate the organisms. Recommendations on how to get accurate VITEK identification results when a blood contaminant is unexpectedly identified as A. vaginae, include using vendor's recommended culture media, assessing microscopic and macroscopic morphologies, and applying alternative identification systems, such as manual identification strips, MALDI-TOF MS or molecular methods to confirm VITEK results.

Impact of different growth media on identification by the VITEK 2 system
All isolates were grown on CBA and Oxy or TSAb and five isolates (21 090, 21 224, 21 216, 21 225 and 21 220) were randomly chosen to be grown on the three media types. Isolates were streaked on agar media and incubated under anaerobic conditions at 37 °C for up to 48 h, until sufficient growth was obtained; isolates derived from each media type were identified with the ANC card using the VITEK 2 system (version 9.01) as per the manufacturer's instructions, with bacterial suspensions prepared in 0.45 % sterile saline solution in polystyrene tubes, and densities verified using the VITEK 2 DENSICHEK apparatus corresponding to MacFarland 3. The ATCC strains of A. vaginae and C. acnes served as controls and were grown on the three media types for 72 and 48 h, respectively.

Identification by alternative methods
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALD-TOF MS) was performed at the Ottawa Hospital reference clinical microbiology laboratory as per the institution's protocols. Briefly, colonies sub-cultured on CDC media were spotted on target plates and identified using MALDI Biotyper RTC. Of the eight PC isolates that were initially identified as A. vaginae, five were chosen for MALDI-TOF MS analysis: two were chosen because these isolates identified as A. vaginae when grown on two of the three media types tested (21 261, 21 266), one identified as C. acnes regardless of the media type tested (21090), and two isolates identified as A. vaginae or C. acnes depending on the media type the suspension was made from (20 047, 21 101) ( Table 1). Three additional PC isolates (21 224, 21 168, 21 202) that were initially identified as C. acnes were included in the MALDI-TOF MS analysis, along with the ATCC A. vaginae isolate that served as a control. Isolates 21 101, 21 090, 21 261 and 21 224 could not be identified using MALDI-TOF MS and were assessed using a primer pair that amplifies a section of the C. acnes 16S rRNA gene previously described by Bernard et al. [9]. The PCR products were run on a 1.5 % agarose gel and the presence of amplicons was assessed. The API RAPID ID 32A strips were used to identify isolates that could not be identified using either the MALDI-TOF MS or PCR methods. Briefly, isolates were sub-cultured on CBA for 48 h to obtain sufficient growth, and suspensions were prepared and dispensed as per the manufacturer's instructions. The isolate that could not be identified using the RAPID strip was then identified using the API 20A strip following subculture on TSAb for 48 h to allow for sufficient growth, after which suspensions were prepared and dispensed as per the manufacturer's instructions.

The microscopic and macroscopic morphology and catalase reaction of A. vaginae is distinguishable from that of C. acnes
The colony and microscopic characteristics and catalase reaction of C. acnes and A. vaginae are shown in Fig. 1. C. acnes produces small white circular convex colonies with regular margins, while A. vaginae produces very small translucent to grey colonies on all three types of media tested. Both species were able to grow more luxuriously on Oxy plates. Microscopically, C. acnes is a Gram-positive pleomorphic rod, while A. vaginae is a Gram-positive short rod-like coccus. C. acnes gives rise to a strong catalase-positive reaction, while A. vaginae is catalase-negative.

Media type affects identification by the VITEK 2 system
The VITEK 2 results obtained for each isolate are listed in Table 1. CDC, Oxy or TSAb were used for initial isolation as per standard procedures implemented at Canadian Blood Services. When isolates were cultured on CBA, TSAb and Oxy for VITEK identification, sufficient growth was obtained more quickly on Oxy plates (as early as 26 h of incubation) compared to the other media. Approximately 76 % of isolates grown on CBA and TSAb were identified as C. acnes, while approximately 23 % of isolates were identified as A. vaginae. Isolates grown on TSAb were 10 % more likely to require further testing (catalase test) to be able to differentiate between the two species when compared to isolates grown on CBA. On the other hand, approximately 38.5 % of isolates grown on Oxy media were identified as A. vaginae. The control ATCC 6919 isolate of C. acnes was identified as C. acnes when grown on TSAb and CBA, but the system could not differentiate between C. acnes and Cutibacterium granulosum when grown on Oxy. Similarly, the control A. vaginae strain was correctly identified when grown on CBA and TSAb but could not be identified when grown on Oxy. A comparison of the biochemical reactions of isolates that had different IDs depending on the agar medium used indicated that the biochemical profiles only differed in three-five reactions, and these differences varied depending on the isolate tested and the level of discrimination obtained. A few of the reactions that distinguished A. vaginae from C. acnes include utilization of arginine (ARG), N-acetyl-d-glucosamine (NAG), d-ribose 2 (dRIB2), and the Ellman's (ELLM) and phenylphosphonate (OPS).

All isolates initially identified as A. vaginae were identified as C. acnes using a multiphasic approach
Four of the eight samples analysed by MALDI-TOF MS were identified as C. acnes (20 047,21 266, 21 168, 21 202). The four isolates that could not be identified were assessed using PCR amplification of a section of the C. acnes 16S RNA gene, but none of these four isolates gave rise to an amplicon as observed for the ATCC C. acnes isolate. The API Rapid ID 32A strip identified three of these isolates as C. acnes (21 101, 21 090, 21 224) while the fourth isolate (21 261) was identified as C. acnes/C. granulosum using the API 20A strip (Table 1).

CONCLUSION
The VITEK 2 system allows for the rapid and accurate identification of clinically relevant bacterial isolates. Anaerobic bacteria together with Corynebacterium species can be identified using the VITEK 2 ANC card. It consists of 36 colorimetric enzymatic reactions, including a combination of biochemical, fermentation, glycosidase and arylamidase tests [10].
Once the card is loaded into the system, the card is assessed for colorimetric changes hourly and the results obtained are matched against an established database. The results of this study demonstrate that the VITEK 2 system can misidentify C. acnes isolates as A. vaginae. Furthermore, the results indicate that the media used to sub-culture bacterial isolates prior to identification may impact on the ability of the VITEK 2 system to accurately identify isolates.
CBA is the supplier-recommended medium to subculture isolates used for QC testing of the ANC cards. Furthermore, supplier recommendations favour relatively fresh anaerobic cultures (24-48 h) for the preparation of the suspension used for identification. Cultures grown on Oxy can grow more rapidly, giving rise to more mature cultures than those grown on CBA or TSAb over the same period of time, which could lead to skewed enzymatic profiles in the ANC card, resulting in the higher proportion of misidentifications. The results obtained in our study suggest that it may be preferable to use CBA for subculture prior to identification, even if Oxy media may appear to be better at promoting the primary growth and isolation of anaerobic bacteria.
Interestingly, a study conducted by Cassity recommended using Oxy plates for identification with VITEK since that media promotes growth of anaerobic bacteria [11]; however, in that study, bacterial identification results were not evaluated. Furthermore, our results highlight the limitations of methods such as MALDI-TOF MS and 16S rRNA amplification (in the absence of sequencing using universal primers [12]) to be able to identify certain isolates. This could potentially be caused by limited databases for reference for MALDI-TOF MS, and primer pair mismatches during PCR amplification in the absence of whole-genome sequences.
Overall, our results illustrate the challenges posed by automated identification of fastidious or slow-growing bacteria such as C. acnes. Misidentification of bacteria, especially in clinical settings, could have serious repercussions for patient treatment. Based on our findings, we recommend having a multiphasic approach for the identification of C. acnes when initial identification with the VITEK system results in a Gram-positive anaerobic bacterium such as A. vaginae, particularly, if it is a bacterium isolated from contaminated transfusable blood components. It is important to consider micro-and macroscopic morphologies, catalase reaction and the use of alternative methods such as MALDI-TOF, if available, or manual identification strips.

Funding information
The study was funded by Canadian Blood Services and Health Canada. The views expressed herein do not necessarily represent those of the Canadian Federal government.
Author contributions C.L. and S.R.-A. conceived the study. Experimental work was done by C.L. and D.K. Manuscript was written by D.K. and reviewed by C.L. and S.R.-A. Five reasons to publish your next article with a Microbiology Society journal 1. When you submit to our journals, you are supporting Society activities for your community.

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Peer review history
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines? Reviewer 1: Yes: Reviewer 1 Comments to Author: This work demonstrates that bacterial misidentification of Cutibacterium acnes as Atopobium vaginae using the VITEK 2 system is most likely caused by the growth medium used for sub-culturing of isolates.
It also describes alternative identification strategies that leads to a better differentiation of the two species.
Although the paper is short, it highlights the importance of a sound assement of doubtful results generated by automated systems like the VITEK 2. line 76-79 -it is stated that the VITEK system was adopted for identification of blood product contaminants in 2019 which the led to the described misidentification. How was the situtation before 2019 regarding the identification of C. acnes? ANSWER: Prior to 2019, microbial identification at Canadian Blood Services was performed with the manual API identification system, and no misidentification of C. acnesas A. vaginaewas reported. This new information has been added in the Introduction of the revised manuscript.
6. Methods line 91 -"...initially misidentified as..." might be better even though the description with "catalase positive" indicates the the PC isolates were wrongly identified ANSWER: Corrected as suggested.
line 106 -based on table 1, all isolates were grown on Columbia agar. It is stated that 5 of the isolates were chosen to be grown on the three media types. 5 isolates of what? If these isolates belong to the initially identified A. vaginae isolates, there are only 4 isolates listed in table 1 that were grown on TSAb. This is a bit confusing, please modify this paragraph for a better clarification.
ANSWER: Table 1 describes only 5 isolates cultured on the three media, Columbia agar, OxyPrass and TSAb (21090, 21224, 21216, 21225, and 21220). This information has been added to the revised manuscript for further clarification in Methods under the subheading "Impact of different growth media on identification by the VITEK system".
line 108 -isolates were grown for up to 72 hours indicating that some isolates were cultivated also for a shorter period dependent on the growth that was obtained. However, as test results of mature cultures might influence the VITEK 2 results, the incubation time is important. Do the authors have any information on the cultivation period of the samples that were used for identification on the VITEK 2 system? ANSWER: Specific incubation times have been updated in Methods un the section "Impact of different growth media on identification by the VITEK system".
line 113 -how is the MALDI measurement performed? any reference to a standard procedure/protocol? ANSWER: Information about the MALDI procedure has been obtained from the Ottawa Hospital and added to Methods under the subheading "Identification by alternate methods". A copy of the hospital's Standard Procedure was sent to us as a reference.

Results and Discussion
line 126-128 -The colony morphology is described briefly. An illustration of the colony morphology in Figure 1 (close-up or enlarged colony) would be preferable. ANSWER: Images have been enlarged equally and have been added to the illustration of the revised Figure 1.
line 149 -There was no Table 2 in section 8. Tables and Figures. Please add the table. ANSWER: This was a typographical error in the original manuscript as there is only one table. It has been corrected in the revised version of the paper. Table 2  ANSWER: We were able to recover the VITEK reports for some of the isolates and confirmed that C. acnesand A. vaginaediffer in only 3-5 reactions. We have added specific information regarding this observation to Results and Discussion under the heading "Media type affect identification by the VITEK 2 system".

Tables and Figures
line 172 -The authors hypothesise that due to a faster growth on OxyPRAS Plus, cultures are more matured leading to a distorted enzymatic profile. An experimentally proof that a shorter incubation time of C. acnes on OxyPRAS Plus results in a better identification would be advisable to support this idea.
ANSWER: We added detailed information obtained during our investigation to the revised manuscript to support our hypothesis. Five isolates (21090, 21224, 21216, 21225, and 21220) were randomly chosen for plating on TSAb, Oxy and CBA media and incubated for up to 48 hrs (see new information in Methods under the subheading "Impact of different growth media on identification by the VITEK system"). As reported in Results and Discussion under the heading "Media type affect identification by the VITEK 2 system", when isolates were cultured on CBA, TSAb and Oxy for VITEK identification, enough growth was obtained fasteron Oxy plates (as early as 26 hrs of incubation), compared to the other media.
As stated in Results and Discussion and shown in Table 1, organisms identified as C. acnesin CBA, were identified as A. vaginaein Oxy plates, providing the experimental proof of misidentification of isolates grown in Oxy compared to other media. We have also added a reference (#11 in the revised manuscript, Cassity TR), which confirms our observations that anaerobic bacteria grow faster on Oxy plates compared to other media.

Methodological rigour
Based on the manuscript, the identification for each isolate was only done once per medium and assay. Can the authors comment on the reproducibility of the results e.g. when an isolate is pre-cultured on one specific media type and tested serveral times on the VITEK 2 system? ANSWER: Each isolate was cultured at least twice prior to identification as per vendor's recommendation. In addition, fresh cultures were prepared for each new VITEK identification.

Presentation of results
No comment.

Literature analysis or discussion
It might be worth to highlight the critical assessment of results generated by automated test system in general at the end of the discussion. Operators in the clinical setting use these systems more and more frequently, however, they need to keep in mind not to trust blindly the results. 1. Methodological rigour, reproducibility and availability of underlying data: The authors need to include more details regarding how they set-up the VITEK ANC card, specifically how was the density of the cell suspension performed and did they follow the vendor's instructions for use (IFU). The IFU incudes precautions and limitations that the authors do not address in their paper such as using only polystyrene tubes and vendor recommended media to name just two. It is also important to include what MALDI-TOF system including database was used to determine identification.
ANSWER: The VITEK ANC card was set up according to vendor instructions, and bacterial suspensions were prepared in polystyrene tubes recommended by the vendor and densities were verified using the VITEK 2 DENSICHEK apparatus. This information has been included in the revised manuscript, in Methods, under the subheading "Impact of different growth media on identification by the VITEK system".
The authors reference table 2 (line 149) however it is not included in the manuscript.
ANSWER: This was a typographical error in the original manuscript, there is only one table, Table 1. The error It has been corrected in the revised version of the paper. Table 1 should include all of the available data to allow readers to easily interpret the study, this includes defining the media that was used in the initial ID, MALDI-TOF, PCR of C acnes 16S rRNA gene, and API 20A strip ID results. See more detailed comments under 5.
ANSWER: Table 1 has been modified. Type of media (table notes) and results from other identification methods have been added as recommended by the Reviewer. Table 1 contains only the VITEK 2 results however the authors compare the strains using other identification methods such as MALDI-TOF, PCR, and API 20A. All of the data should be presented in table 1 to allow the user to better understand the data and the level of misidentification and need for additional testing. See more detailed comments under 5.

Presentation of results:
ANSWER: Table 1 of the revised manuscript includes results of all identification methods.
3. How the style and organization of the paper communicates and represents key findings. The title is misleading as the authors are stating a deficiency of an identification system to correctly identify an organism. The authors show marked improvement in correct identification when using the vendor's recommended media (this needs to be verified as the authors do not confirm that Columbia agar with 5% sheep blood was used). They also present that there is no one perfect identification system, specifically they show a failure to properly identify C. acnes using MALDI-TOF and that PCR amplification of the C acnes 16S rRNA gene failed to provide an identification. Given these facts, it is highly recommended that the title be changed to reflect the full study. A more suitable title would be 'Identification of Cutibacterium acnes during routine bacterial screening of platelet concentrates requires a multiphasic approach'. See more detailed comments under 5.
ANSWER: We thank the Reviewer and accept the suggestion for a revised title to reflect the findings of the study. The title of the revised manuscript summarizes the take home message of our study: "A multiphasic approachto solve misidentification of Cutibacterium acnesas Atopobium vaginaeduring routine bacterial screening of platelet concentrates using the VITEK 2 system". 4. Literature analysis or discussion. The authors should reference the vendor's instruction for use and verify that the correct vendor recommended media (Columbia agar with 5% sheep blood (CBA)) was used. The results of the study do show that the VITEK 2 system may misidentify C acnes as A vaginae however the authors initial results show a higher percentage of misidentification due to using media that is not recommended by the vendor. Using the vendor recommended CBA markedly improves identification.
ANSWER: To clarify, we did use media recommended by the vendor to culture isolates for VITEK identification, including CDC, Oxy (Brucella agar), and TSAb, which the vendor claims give optimal performance (see image below corresponding to Table 2 of package insert for VITEK 2 ANC cards, ref 21347, version 043907-03-2019-03).
Columbia agar is one of the recommended media for test isolates, but it is the recommended media to grow quality control strains. We took a proactive approach during the investigation and tested whether Columbia agar would yield better results (compared to CDC and TSAb) for identification of the test isolates. During improvements of the procedures followed at Canadian Blood Services, CDC plates were replaced with Oxy plates, due to issues of availability of CDC media. Details of media used are found in the revised version of the manuscript, in Methods, under the heading "Agar media".
The results also show that alternative identification systems such as MALDI-TOF and PCR amplification of 16S RNA gene from C acnes does not provide 100% identification and that further confirmation was needed by using API Rapid ID test strips. The authors should conclude that it is important to perform a multiphasic approach for identification of C acnes especially when initial identification using VITEK 2 identifies an anaerobic gram positive organism as A. vaginae. See more detailed comments under 5.
ANSWER: Discussion on the relevance of our findings, including challenges with identification with different approaches, has been added at the end of the Conclusion in the revised manuscript.

Any other relevant comments
Lines 2-3: The title is misleading as the authors are stating a deficiency of an identification system to correctly identify an organism. The authors show marked improvement in correct identification when using the vendor's recommended media. They also present that there is no one perfect identification system, specifically they show a failure to properly identify C. acnes using MALDI-TOF and that PCR amplification of the C acnes 16S rRNA gene failed to provide an identification. Given these facts, it is highly recommended that the title be changed to reflect the full study. A more suitable title would be 'Identification of Cutibacterium acnes during routine bacterial screening of platelet concentrates requires a multiphasic approach ANSWER: We thank the Reviewer and accept the suggestion for a revised title to reflect the findings of the study. The title of the revised manuscript reflects the take home message of our study: "A multiphasic approachto solve misidentification of Cutibacterium acnesas Atopobium vaginaeduring routine bacterial screening of platelet concentrates using the VITEK 2 system". Lines 38-40. The authors should state that MALDI-TOF and PCR amplification of the C acnes 16S rRNA gene were also not able to properly identify C. acnes. The findings from their studies indicate the need for a multiphasic approach for proper identification of this common PC contaminant including using the recommended growth media and supplemental biochemical assays.
ANSWER: This section of the abstract has been edited in the revised manuscript as suggested by the Reviewer.
Line 48. Bold statement. Instead recommend wording as 'to our knowledge we report an issue not reported in the literature concerning misidentification ….' ANSWER: Wording of this section of the impact statement has been changed in the revised manuscript as suggested by the Reviewer.
Line 55. Alternative manual identification systems such as MALDI-TOF and PCR amplification of C acnes16S rRNA , to confirm the VITEK results.
ANSWER: Wording of this section of the impact statement has been changed in the revised manuscript as suggested by the Reviewer.
Lines 58-60.  Lines 69-71. Include the VITEK software version and define the media that is used by CBS to perform the VITEK 2 system identification. The vendor includes performance characteristics including percent misidentification and no identification that can be observed for each software version.
ANSWER: The version of the Vitek software (9.01) used in the study has been added in Methods of the revised manuscript in the section "Impact of different growth media on identification by the VITEK 2 system". The media type used for VITEK identification has been described in a new section "Agar media" in Methods with definitions of anaerobic media in the Introduction of the revised manuscript.
Line 99. Please state if the Columbia agar is with or without blood and if it is with blood please include details, for example with 5% sheep blood and reference it as Columbia blood agar or CBA.
ANSWER: The media used in our study was Columbia agar with 5% sheep blood, and this information has been added in Methods, new section "Agar media" of the revised manuscript.
Line 99. Columbia agar with 5% sheep blood is the vendor recommended media. Please clarify here and throughout the entire manuscript what type of Columbia agar was used. If 5% sheep blood is not included the Columbia agar referenced by the authors is not the vendor recommended media.
ANSWER: Please see above answer to question #4 (literature analysis or discussion). All media used to culture isolates for bacterial identification with the VITEK ANC card (TSAb, CDC, Oxy and CBA) are recommended by the vendor ( Table 2 of the package insert for ANC cards). The Columbia agar media used in our studies was supplemented with 5% sheep blood, as updated in Methods, new section "Agar media" of the revised manuscript.
Line 106. Add descriptor for Columbia agar. Was the agar with or without blood? See comment for page 5, line 99.
ANSWER: As mentioned above, the media used in our study was Columbia agar with 5% sheep blood, and this information has been added in Methods, new section "Agar media" of the revised manuscript.
Line 106. All of the isolates were grown on Columbia agar with 5% sheep blood as recommended by the vendor, while 5 of the isolates were randomly chosen to be grown on all three media types. Note: If Columbia agar did not contain sheep blood it is not the recommended media.
ANSWER: As mentioned above, the media used in our study was Columbia agar with 5% sheep blood, and this information has been added in Methods, new section "Agar media" of the revised manuscript.
Line 109. The authors should confirm if they correctly achieved the vendor recommended density prior to loading the ANC card. How was the density performed? If the authors followed the exact vendor recommended instructions they can reference the vendor instructions for use. The vendor states the importance of cell density, media type, and requirement for using polystyrene tubes in their instructions for use.
ANSWER: The vendor recommendations were followed and densities were verified using a VITEK 2 DENSICHEK apparatus. Details have been added to the revised manuscript in Methods under "Impact of different growth media on identification by the VITEK system".
Line 111. MALDI-TOF is not a molecular method therefore this section should be renamed as Additional identification methods.
ANSWER: The heading of this section has been changed for accuracy to "Identification by alternate methods".
Line 122. Since the authors also relied on API test strips they should include information about how they were used in this section that should be renamed to additional identification methods.
ANSWER: Details on how manual identification was performed has been added to Methods of the revised manuscript in the section entitled "to "Identification by alternate methods" for accuracy".
Lines 125-126. Include a description on how the catalase test was performed. Catalase testing should not be performed from media containing blood as it could lead to false positive results.
ANSWER: Details on how the catalase test was performed has been added to Methods of the revised manuscript in the section entitled "to "Identification by alternate methods" for accuracy".
Line 133, It is important to state what media was used to initially identify the organisms listed in Table 1 under initial ID column. The authors were not using the vendor recommended media for initial identification.
ANSWER: Description of the media used for initial cultures of the isolates has been added in Methods, new section "Agar media".
Line 134. Columbia agar with or without blood (see previous comments). Please clarify.
ANSWER: As mentioned above, the media used in our study was Columbia agar with 5% sheep blood, and this information has been added in Methods, new section "Agar media" of the revised manuscript Lines 136-137. Columbia agar with or without blood (see previous comments). If using the recommended Columbia agar with 5% sheep blood the authors should further clarify by stating '… grown on the vendor recommended CBA'. ANSWER: As mentioned above, the media used in our study was Columbia agar with 5% sheep blood, and this information has been added in Methods, new section "Agar media" of the revised manuscript. Columbia agar with 5% sheep blood is not the only recommended media by the vendor to grow test isolates.
Line 142. The media study clearly shows the importance of using the vendor recommended culture media for identification using the VITEK 2 system. This result confirms the precaution listed in the vendor instructions for use that 'use of culture media other than the recommended types must be validated by the customer laboratory for acceptable performance'. ANSWER: Please see above answer to question #4 (literature analysis or discussion). All media used to culture isolates for bacterial identification with the VITEK ANC card (TSAb, CDC, Oxy and CBA) are recommended by the vendor. The Columbia agar media used in our studies is supplemented with 5% sheep blood, as updated in Methods, new section "Agar media" of the revised manuscript.
Line 106. Add descriptor for Columbia agar. Was the agar with or without blood? See comment for page 5, line 99.
ANSWER: As mentioned above, the media used in our study was Columbia agar with 5% sheep blood, and this information has been added in Methods, new section "Agar media" of the revised manuscript.
Line 143. This subtitle is misleading. All of the isolates were not identified as C acnes by just one method. Full identification required a multiphasic approach. The authors used MALDI-TOF, PCR amplification of a section of the C acnes 16S RNA gene as well as using API Rapid ID 32A strip testing to confirm proper identification. This section should be renamed as 'Multiple alternative identification tests were required to identify all isolates initially identified as A vaginae as C acnes.' ANSWER: The heading of this section has been changed to "All isolates initially identified as A. vaginaewere identified as C. acnesusing a multiphasic approach" for accuracy.
ANSWER: Table 1 of the revised manuscript includes results of all identification methods.
Line 149. Table 2 is missing from the manuscript. Recommend adding API results to Table 1. Table 1 should provide all of the data together so readers are able to easily analyze the data in order to better illustrate the importance of using vendor recommended media and the need for additional testing to provide proper identification.
ANSWER: Table 2 does not exist, and the error has been fixed in the revised version of the manuscript. Table 1 of the revised manuscript includes results of all identification methods.
Lines 170. ….for quality control and routine testing of the ANC cards. Reference Table 2 in the vendor's instruction for use. Table  2 in the instructions for use provides a list of the media that were used in the identification product database development and that will give the optimal performance. ANSWER: Please see above answer to question #4 (literature analysis or discussion). We used information of Table 2 of the package insert of the ANC card to support the experimental approach of our investigation. All media used to culture isolates for bacterial identification with the VITEK ANC card (TSAb, CDC, Oxy and CBA) are recommended by the vendor. The Columbia agar media used in our studies is supplemented with 5% sheep blood, as updated in Methods, new section "Agar media" of the revised manuscript.
Lines 166-170. The results of the study do show that the VITEK 2 system may misidentify C acnes as A vaginae however the authors initial results show a higher percentage of misidentification due to using media that is not recommended by the vendor. Using the vendor recommended CBA markedly improves identification. The results also show that alternative identification systems such as MALDI-TOF and PCR amplification of 16S RNA gene from C acnes does not provide 100% identification and that further confirmation was needed by using API Rapid ID test strips. The authors should conclude that it is important to perform a multiphasic approach for identification of C acnes especially when initial identification using VITEK 2 identifies an anaerobic gram positive organism as A. vaginae.
Line 205. Recommend referencing the vendor instructions for use for the VITEK 2 system. ANSWER: Information has been added at the end of the Conclusion of the revised manuscript addressing the Reviewer's comment.

VERSION 1
Editor recommendation and comments https://doi.org/10.1099/acmi.0.000539.v1.5 Comments: 1. Methodological rigour, reproducibility and availability of underlying data: The authors need to include more details regarding how they set-up the VITEK ANC card, specifically how was the density of the cell suspension performed and did they follow the vendor's instructions for use (IFU). The IFU incudes precautions and limitations that the authors do not address in their paper such as using only polystyrene tubes and vendor recommended media to name just two. It is also important to include what MALDI-TOF system including database was used to determine identification. The authors reference table 2 (line 149) however it is not included in the manuscript. Table 1 should include all of the available data to allow readers to easily interpret the study, this includes defining the media that was used in the initial ID, MALDI-TOF, PCR of C acnes 16S rRNA gene, and API 20A strip ID results. See more detailed comments under 5. 2. Presentation of results: Table 1 contains only the VITEK 2 results however the authors compare the strains using other identification methods such as MALDI-TOF, PCR, and API 20A. All of the data should be presented in table 1 to allow the user to better understand the data and the level of misidentification and need for additional testing. See more detailed comments under 5. 3. How the style and organization of the paper communicates and represents key findings. The title is misleading as the authors are stating a deficiency of an identification system to correctly identify an organism. The authors show marked improvement in correct identification when using the vendor's recommended media (this needs to be verified as the authors do not confirm that Columbia agar with 5% sheep blood was used). They also present that there is no one perfect identification system, specifically they show a failure to properly identify C. acnes using MALDI-TOF and that PCR amplification of the C acnes 16S rRNA gene failed to provide an identification. Given these facts, it is highly recommended that the title be changed to reflect the full study. A more suitable title would be 'Identification of Cutibacterium acnes during routine bacterial screening of platelet concentrates requires a multiphasic approach'. See more detailed comments under 5. 4. Literature analysis or discussion. The authors should reference the vendor's instruction for use and verify that the correct vendor recommended media (Columbia agar with 5% sheep blood (CBA)) was used. The results of the study do show that the VITEK 2 system may misidentify C acnes as A vaginae however the authors initial results show a higher percentage of misidentification due to using media that is not recommended by the vendor. Using the vendor recommended CBA markedly improves identification. The results also show that alternative identification systems such as MALDI-TOF and PCR amplification of 16S RNA gene from C acnes does not provide 100% identification and that further confirmation was needed by using API Rapid ID test strips. The authors should conclude that it is important to perform a multiphasic approach for identification of C acnes especially when initial identification using VITEK 2 identifies an anaerobic gram positive organism as A. vaginae. See more detailed comments under 5. 5. Any other relevant comments Lines 2-3: The title is misleading as the authors are stating a deficiency of an identification system to correctly identify an organism. The authors show marked improvement in correct identification when using the vendor's recommended media. They also present that there is no one perfect identification system, specifically they show a failure to properly identify C. acnes using MALDI-TOF and that PCR amplification of the C acnes 16S rRNA gene failed to provide an identification. Given these facts, it is highly recommended that the title be changed to reflect the full study. A more suitable title would be 'Identification of Cutibacterium acnes during routine bacterial screening of platelet concentrates requires a multiphasic approach Line 22: add multiphasic, MALDI-TOF, 16S RNA to keywords Lines 38-40. The authors should state that MALDI-TOF and PCR amplification of the C acnes 16S rRNA gene were also not able to properly identify C. acnes. The findings from their studies indicate the need for a multiphasic approach for proper identification of this common PC contaminant including using the recommended growth media and supplemental biochemical assays. Line 48. Bold statement. Instead recommend wording as 'to our knowledge we report an issue not reported in the literature concerning misidentification ….' Line 55. Alternative manual identification systems such as MALDI-TOF and PCR amplification of C acnes16S rRNA , to confirm the VITEK results. Lines 58-60. Table 1 needs to include both MALDI-TOF and molecular results. Authors may reference outside supporting laboratories in the table. The table should also reference what media was used to generate the initial ID. Line 67. Define the type of anaerobic agar. Lines 69-71. Include the VITEK software version and define the media that is used by CBS to perform the VITEK 2 system identification. The vendor includes performance characteristics including percent misidentification and no identification that can be observed for each software version. Line 99. Please state if the Columbia agar is with or without blood and if it is with blood please include details, for example with 5% sheep blood and reference it as Columbia blood agar or CBA. Line 99. Columbia agar with 5% sheep blood is the vendor recommended media. Please clarify here and throughout the entire manuscript what type of Columbia agar was used. If 5% sheep blood is not included the Columbia agar referenced by the authors is not the vendor recommended media. Line 106. Add descriptor for Columbia agar. Was the agar with or without blood? See comment for page 5, line 99. Line 106. All of the isolates were grown on Columbia agar with 5% sheep blood as recommended by the vendor, while 5 of the isolates were randomly chosen to be grown on all three media types. Note: If Columbia agar did not contain sheep blood it is not the recommended media. Line 109. The authors should confirm if they correctly achieved the vendor recommended density prior to loading the ANC card. How was the density performed? If the authors followed the exact vendor recommended instructions they can reference the vendor instructions for use. The vendor states the importance of cell density, media type, and requirement for using polystyrene tubes in their instructions for use. Line 111. MALDI-TOF is not a molecular method therefore this section should be renamed as Additional identification methods. Line 122. Since the authors also relied on API test strips they should include information about how they were used in this section that should be renamed to additional identification methods. Lines 125-126. Include a description on how the catalase test was performed. Catalase testing should not be performed from media containing blood as it could lead to false positive results. Line 133, It is important to state what media was used to initially identify the organisms listed in Table 1 under initial ID column. The authors were not using the vendor recommended media for initial identification. Line 134. Columbia agar with or without blood (see previous comments). Please clarify. Lines 136-137. Columbia agar with or without blood (see previous comments). If using the recommended Columbia agar with 5% sheep blood the authors should further clarify by stating '… grown on the vendor recommended CBA'. Line 142. The media study clearly shows the importance of using the vendor recommended culture media for identification using the VITEK 2 system. This result confirms the precaution listed in the vendor instructions for use that 'use of culture media other than the recommended types must be validated by the customer laboratory for acceptable performance'. Line 143. This subtitle is misleading. All of the isolates were not identified as C acnes by just one method. Full identification required a multiphasic approach. The authors used MALDI-TOF, PCR amplification of a section of the C acnes 16S RNA gene as well as using API Rapid ID 32A strip testing to confirm proper identification. This section should be renamed as 'Multiple alternative identification tests were required to identify all isolates initially identified as A vaginae as C acnes.' Lines 145-147. Include the MALDI-TOF and 16S rRNA results in Table 1. Line 149. Table 2 is missing from the manuscript. Recommend adding API results to Table 1. Table 1 should provide all of the data together so readers are able to easily analyze the data in order to better illustrate the importance of using vendor recommended media and the need for additional testing to provide proper identification. Lines 170. ….for quality control and routine testing of the ANC cards. Reference Table 2 in the vendor's instruction for use. Table 2 in the instructions for use provides a list of the media that were used in the identification product database development and that will give the optimal performance. Lines 166-170. The results of the study do show that the VITEK 2 system may misidentify C acnes as A vaginae however the authors initial results show a higher percentage of misidentification due to using media that is not recommended by the vendor. Using the vendor recommended CBA markedly improves identification. The results also show that alternative identification systems such as MALDI-TOF and PCR amplification of 16S RNA gene from C acnes does not provide 100% identification and that further confirmation was needed by using API Rapid ID test strips. The authors should conclude that it is important to perform a multiphasic approach for identification of C acnes especially when initial identification using VITEK 2 identifies an anaerobic gram positive organism as A. vaginae. Line 205. Recommend referencing the vendor instructions for use for the VITEK 2 system.

Please rate the manuscript for methodological rigour Satisfactory
Please rate the quality of the presentation and structure of the manuscript Satisfactory